Dendritic co-stratification of ON and ON-OFF directionally selective ganglion cells with starburst amacrine cells in rabbit retina.

The morphology, dendritic branching patterns, and dendritic stratification of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These cells have been characterized in the preceding paper in terms of their cell body size, dendritic field size, and branching pattern. In this paper, the two kinds of DS ganglion cell are compared in terms of their levels of dendritic stratification. They are compared with each other and also with examples of class III.1 cells, defined in the preceding paper with reference to our previous studies. Studies employing computer-aided, 3D reconstruction of dendritic trees, as well as analysis of a pair of ON DS and ON-OFF DS ganglion cells with overlapping dendritic trees show that the two types of DS ganglion cell partly co-stratify in the middle of sublamina b (stratum 4). The report that some ON DS ganglion cells extend a few dendrites into sublamina a is confirmed. The study of pairs of ON-OFF DS ganglion cells and starburst amacrine cells with overlapping dendritic trees reveals a precise co-stratification of these two cell types, and many points of close apposition of starburst boutons with ON-OFF DS ganglion cell dendrites in both sublaminae of the inner plexiform layer (IPL). This is confirmed by high-resolution light microscopy and by electron microscopy. It is possible to conclude, therefore, that ON DS are also partly co-stratified with type b starburst (cholinergic) amacrine cells, and are apparently also partly co-stratified with type a starburst amacrine cells, when occasional dendrites rise to that level. The co-stratification of the two kinds of DS ganglion cell is consistent with the sharing of some inputs in common, including some cone bipolar cell inputs. The co-stratification of both with starburst amacrine cells agrees with the physiological demonstration of the powerful pharmacological effects upon ON and ON-OFF DS ganglion cells reported for cholinergic agonists. The major difference in the dendritic stratification of bistratified ON-OFF DS ganglion cells and generally unistratified ON DS ganglion cells is consistent with the bisublaminar organization of ON and OFF pathways in the IPL. The problem of occasional branches of ON DS cells in sublamina a is discussed in terms of a threshold for OFF responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic organization of complex ganglion cells in rabbit retina: type and arrangement of inputs to directionally selective and local-edge-detector cells

The type and topographic distribution of synaptic inputs to a directionally selective (DS) rabbit retinal ganglion cell (GC) were examined and were compared with those received by two other complex GC types. The percentage of cone bipolar cell (BC) input, presumably an index of sustained responses and simple receptive field properties, is much higher than expected for complex GCs in reference to previous reports in other species: approximately 20% for the type 1 bistratified ON-OFF DS GC and for a multistratified GC, and approximately 40% for the small-tufted local-edge-detector GC. Consistent with a previous study (Famiglietti [1991] J. Comp. Neurol. 309:40-70), no ultrastructural evidence is found for inhibitory synapses from starburst amacrine cells to the ON-OFF DS GC. The density of inputs to the ON-OFF DS GC is high and rather evenly distributed over the dendritic tree. Clustering of inputs brings excitatory and inhibitory inputs into proximity, but the strict on-path condition of more proximal inhibitory inputs, favoring shunting inhibition, is not satisfied. Prominent BC input and its regional variation suggest that BCs play key roles in DS neural circuitry, both pre- and postsynaptic to the ON-OFF DS GC, according to a bilayer model (Famiglietti [1993] Invest. Ophthalmol. Vis. Sci. 34:S985). Asymmetry of inhibitory amacrine cell input may signify a region on the preferred side of the receptive field, the inhibition-free zone (Barlow and Levick [1965] J. Physiol. (Lond.) 178:477-504), supporting a role for postsynaptic integration in the DS mechanism. Prominent BC input to the local-edge-detector, often without accompanying amacrine cell input, indicates presynaptic integration in forming its trigger feature.     

Synaptic input to the on-off directionally selective ganglion cell in the rabbit retina

A physiologically identified on-off directionally selective (DS) ganglion cell with its preferred-null axis defined was stained with horseradish peroxidase (HRP) and prepared for electron microscopy. A continuous series of thin sections were used to examine the cell's synaptology. Although the DS cell dendrite received the majority of its synaptic input from a heterogeneous population of amacrine cell processes, a frequently observed synaptic profile consisted of a DS cell dendrite receiving synapses from a cluster of several amacrine cell processes. These clusters of processes were assumed to be from a fascicle of amacrine cells, most of which probably belonged to several different cholinergic starburst amacrine cells. The most frequently observed presynaptic profile within the clusters consisted of a synaptic couplet in which two processes synapsed with each other before one of them finally synapsed with the DS ganglion cell dendrite; occasionally, a chain of three serial synapses was seen. In addition, a specific microcircuit that has the potential to exert lateral feedforward inhibition was also observed. This microcircuit consisted of two cone bipolar cell terminal dyad synapses where one dyad contained an amacrine cell process making a reciprocal synapse and a DS ganglion cell dendrite receiving direct excitation; the other dyad synapse, found lateral to the first dyad, contained two amacrine cell processes that both made reciprocal synapses, but one fed forward to make a putative inhibitory synapse with the DS cell dendrite.     

Polyaxonal amacrine cells of rabbit retina: size and distribution of PA1 cells.

Type 1 polyaxonal (PA1) amacrine cells have been identified previously in rabbit retina, and their morphological characteristics have been described in detail in the preceding paper. Like other polyaxonal amacrine cells they bear distinct dendritic and axonal branching systems, the latter of which originates in two to six thin, branching axons which emerge from or near to the cell body. Unlike other types of polyaxonal amacrine cells, however, their branching is stratified at the a/b sublaminar border and their cell bodies are most often displaced interstitially in the inner plexiform layer (IPL). This report emphasizes quantitative features of the population of PA1 cells, documented in Golgi-impregnated and Nissl-stained retinas, and provides further evidence in Nissl preparations for the amacrine-cell nature of polyaxonal amacrine cells. The cell bodies of Golgi-impregnated PA1 amacrine cells are relatively large: 12-15 microns in equivalent diameter over the range extending from the visual streak 6 mm into ventral retina. Over the same range, dendritic trees are 400-800 microns in equivalent diameter, but they are much smaller than the axonal arborizations, which extend up to and perhaps beyond 2 mm from the cell body. Interstitial cell bodies appropriate to PA1 cells have been identified in Nissl-stained, whole-mounted rabbit retinas. In the plane of the retina, these are comparable in area to smaller medium-size ganglion cells, but their very pale Nissl staining, high nuclear/cytoplasmic ratio, and absence of nucleolar staining are all characteristics of amacrine cells. Interstitial displacement of presumed PA1 cells is rare in the visual streak, and the frequency of interstitial cells reaches a peak between 1 and 2 mm ventral to the streak. Counts in Nissl-stained retinas and estimates from nearest neighbor analyses in these and in Golgi-impregnated retinas indicate a density of PA1 cells in the range of 15-16 cells/mm2 at about 2 mm ventral to the streak, when an estimated 25% shrinkage of the material is taken into account. Dendritic field overlap, based upon this estimate, is calculated to be about fourfold, while a lower bound to estimates of the overlap of axonal arborizations is nearly an order of magnitude higher. Many similarities are noted in a qualitative and quantitative comparison of PA1 amacrine cells in rabbit and monkey retinas. In assessing the contribution of the structural organization of PA1 amacrine cells to their possible functional role(s), it is notable that their appearance conforms not to amacrine cells as commonly viewed, but to a more conventional model of neuronal dynamic polarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

‘Starburst’ amacrine cells and cholinergic neurons: mirror-symmetric ON and OFF amacrine cells of rabbit retina

Golgi-impregnated 'starburst' amacrine cells share significant morphological features with cholinergic neurons in rabbit retina. They are mirror-symmetrical about the a/b (OFF/ON) sublaminar border of the inner plexiform layer. Type a starburst amacrines have cell bodies in the amacrine cell layer and dendrites in sublamina a, while type b cells have their cell bodies in the ganglion cell layer and dendrites in sublamina b of the inner plexiform layer (IPL). The two levels of narrow dendritic stratification are precisely those demonstrated by Masland and Mills for cholinergic amacrine cells. The morphological evidence indicates that the duality of ON and OFF pathways is served separately by type b (displaced) and type a starburst amacrine cells, respectively.     

Synaptic organization of neurotensin immunoreactive amacrine cells in the chicken retina

Immunohistochemistry was utilized to investigate the light and electron microscopic localization of neurotensinlike immunoreactive (NT) amacrine cells in the chicken retina. The NT cells possess oval cell bodies (7 microns in diameter) that are located in either the second or third tier of cells from the border of the inner nuclear and inner plexiform layers. The processes of such cells extend into the inner plexiform layer where they ramify as a narrow plexus in sublamina 1 and as a broad plexus in sublaminas 3 and 4. Additionally, stained processes are observed occasionally within sublamina 5. At the ultrastructural level, NT-positive somas exhibit a rather dense and evenly distributed peroxidase reaction product throughout their cytoplasm. The nucleus of NT amacrine cells possess a round, unindented nuclear membrane. NT-immunoreactive processes in the inner plexiform layer interact synaptically only with non-NT cells. NT processes receive synaptic input mainly from the processes of amacrine cells and to a lesser degree from bipolar cells. The large majority of NT-stained varicosities form presynaptic contacts onto the processes of amacrine cells, but are also presynaptic to bipolar cell axon terminals. Moreover, each of the above synaptic relationships can be identified in each of sublaminas 1 and 3 to 4 of the inner plexiform layer. In addition, NT processes are presynaptic to processes devoid of synaptic vesicles that may originate from ganglion cells. Finally, NT processes occasionally form synaptic contacts onto somas situated in the most proximal row of the inner nuclear layer.     

Dendritic maturation of displaced putative cholinergic amacrine cells in the rabbit retina

The dendritic trees of Cb, cholinergic, amacrine cells in the ganglion cell layer of the developing rabbit retina are revealed by intracellular injection with Lucifer yellow to have the adult dendritic branching pattern at birth. It is demonstrated that these cells maintain a constant number of dendritic branches throughout postnatal development and that their dendritic trees increase in size by the growth and subsequent elongation of all branches. Proximal and distal dendrites increase in length by almost the same proportions between birth and adulthood. Although the adult pattern of dendritic branching of Cb amacrine cells is established by birth, dendrites in the young possess numerous short appendages (1-5 microns in length) resembling the "dendritic spines" of immature cat retinal ganglion cells. Some of these structures remain on the dendrites of adult cells but the majority are lost at the end of the third postnatal week. As dendritic spines disappear, the dendrites of Cb amacrine cells, especially the distal portion of the tree, acquire numerous varicosities. At each stage after P10, the gain in the number of varicosities greatly exceeds the loss in spines; this is not consistent with the hypothesis that all varicosities are retracted dendritic spines. The rapid increase in the number of varicosities on distal dendrites of Cb amacrine cells during the first 3 postnatal weeks coincides with the maturation of amacrine cell physiological responses. There is no distinct centroperipheral gradient in the postnatal dendritic maturation (acquisition of varicosities, loss of spines, attainment of the adult number of branches) of Cb amacrine cells from the visual streak to the peripheral retina. However, the area of their dendritic tree increases relatively more in the retinal periphery compared to that in the visual streak.     

Polyaxonal amacrine cells of rabbit retina: morphology and stratification of PA1 cells.

Polyaxonal amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes, closely resembling the axons of Golgi type II cells found elsewhere in the central nervous system. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, three have been described previously in brief communications, and one is the subject of this paper. Type 1 polyaxonal (PA1) amacrine cells have larger cell bodies than most amacrine cells in Golgi preparations, averaging about 13 microns in diameter. These are typically positioned interstitially in the middle of the inner plexiform layer (IPL), although some are also found in the amacrine and ganglion cell layers. Axons and dendrites are broadly stratified in the middle of the IPL, in the vicinity of the a/b sublaminar border. Sparsely branching dendrites have a conventional appearance, branching at a narrow angle, and giving rise to smaller daughter branches, which taper gradually toward their termination. An unusual feature of the dendrites is the zig-zag course of some terminal branches. Clusters of small, pedunculated spines are common on proximal dendrites, and spines are virtually absent on axons. Axons emerge from proximal dendrites within 50 microns of the soma, and more rarely from the soma, in a tapering initial segment, commonly interrupted by one or two large swellings. Subsequent branching is at a wide angle, and the fine caliber is maintained in the transition from parent to daughter branches. The uniform thickness of the axonal branches is interrupted at intervals by boutons en passant. Although the extent of the dendritic tree is large, exceeding 500 microns in radial extent from the cell body, for cells a few millimeters distant from the visual streak, the axonal tree is much larger, and its radial extent is measured in millimeters. PA1 amacrine cells are believed to be polarized in their functional organization, with a primarily recipient dendritic tree and a primarily transmissive axonal tree. PA1 amacrine cells co-stratify with nab cone bipolar cells and with certain small tufted amacrine and ganglion cells at the a/b sublaminar border. The co-stratification of both axons and dendrites at the a/b sublaminar border of the IPL suggests that PA1 amacrine cells are important modulators of neural activity in the middle of the IPL, affecting both ON and OFF responses, and perhaps ON-OFF cells selectively.     

Morphology and physiology of the polyaxonal amacrine cells in the rabbit retina.

We examined the morphology and physiological response properties of the axon-bearing, long-range amacrine cells in the rabbit retina. These so-called polyaxonal amacrine cells all displayed two distinct systems of processes: (1) a dendritic field composed of highly branched and relatively thick processes and (2) a more extended, often sparsely branched axonal arbor derived from multiple thin axons emitted from the soma or dendritic branches. However, we distinguished six morphological types of polyaxonal cells based on differences in the fine details of their soma/dendritic/axonal architecture, level of stratification within the inner plexiform layer (IPL), and tracer coupling patterns. These morphological types also showed clear differences in their light-evoked response activity. Three of the polyaxonal amacrine cell types showed on-off responses, whereas the remaining cells showed on-center responses; we did not encounter polyaxonal cells with off-center physiology. Polyaxonal cells respected the on/off sublamination scheme in that on-off cells maintained dendritic/axonal processes in both sublamina a and b of the IPL, whereas processes of on-center cells were restricted to sublamina b. All polyaxonal amacrine cell types displayed large somatic action potentials, but we found no evidence for low-amplitude dendritic spikes that have been reported for other classes of amacrine cell. The center-receptive fields of the polyaxonal cells were comparable to the diameter of their respective dendritic arbors and, thus, were significantly smaller than their extensive axonal fields. This correspondence between receptive and dendritic field size was seen even for cells showing extensive homotypic and/or heterotypic tracer coupling to neighboring neurons. These data suggest that all polyaxonal amacrine cells are polarized functionally into receptive dendritic and transmitting axonal zones.     

Development of cholinergic amacrine cells is visual activity-dependent in the postnatal mouse retina

In the present study, we used immunocytochemistry to study the temporal and spatial arrangement of mouse cholinergic amacrine cells during postnatal retinal development under normal light/dark cycles and during visual deprivation. Choline acetyltransferase (ChAT)-immunolabeled cells were detected in the neuroblastic layer (NBL) and in the ganglion cell layer (GCL) at postnatal day 0 (P0). Between P3-5, two characteristic cholinergic bands were clearly identified in the inner plexiform layer (IPL). The signal intensity of somas and processes progressively increased over the first 2 postnatal weeks. Around eye opening at P12, cholinergic neurons were mature-like. This early developmental process was not altered by visual deprivation. After eye opening, the space between the two cholinergic bands increased continuously and the spatial regularity index changed constantly, indicating that the cholinergic neurons possibly underwent refinement during later postnatal development. The changes occurring following eye opening were retarded by visual deprivation. The morphologies of photoreceptors, horizontal cells, recoverin-positive OFF-cone bipolar cells, rod bipolar cells, dopaminergic amacrine cells, and Müller cells appeared normal. Their stratification in the outer plexiform layer (OPL) and the IPL was not affected by visual deprivation. However, glial cells grew vertically across the entire thickness of dark-reared retinas. Our results suggest that the development of cholinergic neurons before eye opening is independent of the lighting conditions. Their development after eye opening is greatly impeded by visual deprivation. This visual activity-dependent phase of development may be a critical period for the maturation and synaptic wiring of cholinergic amacrine cells in the mammalian retina.     

Polyaxonal amacrine cells of rabbit retina: PA2, PA3, and PA4 cells. Light and electron microscopic studies with a functional interpretation.

Polyaxonal (PA) amacrine cells are a new class of amacrine cell bearing one to six branching, axon-like processes that emerge from the cell body or dendritic trees within 50 microns of the cell body. These slender processes of uniform caliber branch at right angles and in many respects closely resemble the axons of Golgi type II cells found elsewhere in the brain. Of the four types of polyaxonal amacrine cell that we have recognized in rabbit retina, two have been described previously in brief communications. One of these, the PA1 amacrine cell with its interstitially displaced cell body, located in the inner plexiform layer (IPL), has been analyzed extensively in two preceding reports. This paper concerns PA2, PA3, and PA4 amacrine cells. Type 2 polyaxonal (PA2) amacrine cells, identified in Golgi preparations of whole-mounted rabbit retinas, have smaller cell bodies (9-14 microns) than the other three types and these are always displaced to the ganglion cell layer (GCL) or the inner border of the inner plexiform layer (IPL). The dendritic fields of PA2 cells are also smaller than those of other PA amacrine cells, and most of their sparse dendritic branching is narrowly stratified at the border of strata (S) 4 and 5. Some members of this more heterogeneous amacrine cell "type" are bistratified, however, and more highly branched with terminal branches rising to end in S1. PA2 amacrine cells bear a scattering of small dendritic spines and may also exhibit complex dendritic appendages arising at the ends of terminal branches in proximal regions of the dendritic tree. PA2 cells emit one to three axons from the proximal dendritic tree, and about half of the cells bear a single axon. Type 3 polyaxonal (PA3) amacrine cells resemble PA1 cells in the large size of their cells bodies (11-16 microns) and dendritic fields, but differ from the latter in placement of cell bodies, which is in the GCL, and dendritic and axonal stratification, which is multistratified, ranging from S4 to S1, with a concentration in S3 or S4 and a variable contribution to S1. PA3 cells differ from PA1 cells in several other respects, including dendritic branching which occurs at higher frequency and is biased toward temporal retina, and in characteristic bristling dendritic spines, clustered in the intermediate regions of the dendritic tree, that are longer, more variable in appearance and more tightly clustered than the small, uniform spines of PA1 cells that are clustered on proximal dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail. With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-microm-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 microm. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina.     

Postnatal development of tyrosine hydroxylase immunoreactive amacrine cells in the rabbit retina: II. Quantitative analysis.

Tyrosine hydroxylase (TH)-immunoreactive (IR) amacrine cells of the rabbit retina mature during the first four postnatal weeks, and their cellular development is described in the preceding paper (Casini, G., and N.C. Brecha, J. Comp. Neurol. 326:283-301, 1992). The present investigation is a quantitative analysis of the postnatal development of the TH-IR amacrine cell population. TH-IR amacrine cells gradually increase in size from birth (soma area of 44.7 +/- 12.4 microns2, mean +/- standard deviation) to adulthood (144.2 +/- 28.0 microns2). Cell density slightly increases from postnatal day (PND) 0 (41.9 +/- 9.5 cells/mm2) to PND 6 (47.2 +/- 7.2 cells/mm2), then markedly decreases from PND 6 to adulthood (17.8 +/- 5.3 cells/mm2) as a consequence of retinal growth. TH-IR cell number almost doubles from PND 0 (about 4,100 cells/retina) to adulthood (about 7,850 cells/retina). The increase in the total number of TH-IR amacrine cells can be explained by the generation of new TH-IR cells in the inner nuclear layer, a delay in the expression of the TH phenotype after neurogenesis by cells committed to be dopaminergic, or the acquisition of this dopaminergic phenotype by uncommitted cells. The development of the TH-IR amacrine cell mosaic was assessed by an evaluation of the distribution of nearest neighbor distances of TH-IR cells. There is a poor correlation between this distribution and a theoretical nonrandom distribution before PND 12. After this age, the nearest neighbor distance distribution shifts towards a nonrandom distribution, and is similar to that of the TH-IR amacrine cell population in the adult retina. The establishment of the TH-IR amacrine cell population mosaic is likely to be achieved through different interacting events, including intrinsic (e.g., genetic) factors, environmental influences, and nonuniform retinal growth. Overall, the population parameters analyzed in the present study approach adult values about the time of eye opening (PND 12) and they reach adult values by PND 26.     

A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections

To observe certain quantitative features of neuronal geometry and microcircuitry, it is necessary to reconstruct neurons from electron micrographs of serial, ultrathin sections. We describe here an approach to preparing, photographing, and analyzing moderately long series (100–500 sections). A series is prepared using an assembly line approach: one operator cuts while a second mounts ribbons of sections using various mechanical aids. Photographs are taken in the electron microscope at low magnification and high accelerating voltage. Sequential negatives are aligned using an image combiner and copied, using quasi-coherent illumination, onto 35 mm film. The resulting ‘movie’ is mounted on a precision film transport mounted on an X-Y stage controlled by stepping motors. The movie is viewed through a high resolution video system while a video storage device and switching system permit rapid alternation between frames for comparisons. The profiles of a process in successive frames are ‘microaligned’ by small adjustments of the transport's X-Y position. The absolute X-Y biological coordinates for each frame and the correction necessary to bring it into alignment are stored in a Z80 microprocessor as a process vector. When the movie is re-examined with the stepping motors under control of the computer, the microaligned process shows almost no frame-to-frame jitter. The process vector may be used to generate a ‘branch schematic’ of the neuron. The microaligned profiles can also be digitized and displayed as a reconstruction using a PDF 11/34 computer. Uses of the approach are presented with examples from the cat retina and visual cortex.     

Synaptic connections of starburst amacrine cells and localization of acetylcholine receptors in primate retinas

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.     

Characterization of small-field bistratified amacrine cells in macaque retina labeled by antibodies against synaptotagmin-2

Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin‐2 (Syt2). Syt2 was localized in a population of small‐field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF‐layer and only a few lobular processes extending into the ON‐layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2‐labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ‐aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ～200/mm² in peripheral retina to ～1,400/mm² in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2‐labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small‐field amacrine cells closely resembling A8 amacrine cells and their cone‐dominated bipolar cell input. J. Comp. Neurol. 521:709–724, 2013. © 2012 Wiley Periodicals, Inc.     

Midget ganglion cells of the parafovea of the human retina: a study by electron microscopy and serial section reconstructions.

In this study we used serial section electron microscopy and three-dimensional reconstructions to examine four midget ganglion cells of the human retina. The four cells were located in the parafoveal retina 2.5 mm or 8 degrees from the foveal center. Both type a (with dendritic trees in distal inner plexiform layer) and type b (with dendritic trees in proximal inner plexiform layer) midget ganglion cells have been studied. These cells have dendritic trees of 7-9 microns diameter, and their complete dendritic trees in the neuropil of the inner plexiform layer can be analyzed, as well as the bipolar cell axon terminals having synaptic input, by a study of 100-150 serial ultrathin sections. Type a midget ganglion cells appear to be in a one-to-one relationship with flat midget bipolar cell axon terminals ending in distal inner plexiform layer. Type b midget ganglion cells are in a one-to-one synaptic relationship with invaginating midget bipolar cell axon terminals in proximal inner plexiform layer. The midget bipolar cells primarily involved with the midget ganglion cells do not contact other ganglion cell dendrites. In other words, midget bipolar cells appear to be in exclusive contact with single midget ganglion cells in the human retina. The midget ganglion cells receive most of their input from their associated midget bipolar cells in the form of ribbon synapses at dyads or monads (55-81 ribbons total), although ribbonless synapses are seen occasionally. In all four midget ganglion cells reconstructed, one or two other bipolar cell axon terminals, presumed to be from wide-field bipolar types, provide 1-3 ribbon synapses each. The number of amacrine synapses upon a midget ganglion cell's dendritic tree is approximately equal to the number of bipolar ribbon inputs (43%-56% bipolar ribbons: 44%-57% amacrine synapses). We assume from our knowledge of response characteristics of ganglion cells in other mammalian retinas (Nelson et al., '78: J. Neurophysiol. 41:427-483), that the type a midget ganglion cell and its exclusive connectivity with a flat midget bipolar cell forms a single cone connected OFF-center pathway, whereas the type b midget ganglion cell with its exclusive connectivity to an invaginating midget bipolar cell forms a single cone connected ON-center pathway, through the retina to the brain.     

Postnatal development of tyrosine hydroxylase immunoreactive amacrine cells in the rabbit retina: I. Morphological characterization.

The present and accompanying (Casini, G., and N.C. Brecha, J. Comp. Neurol. 326:302-313, 1992) papers investigate the postnatal development of tyrosine hydroxylase (TH)-immunoreactive (IR) amacrine cells in the rabbit retina. This study is focused on a detailed analysis of the patterns of cellular growth and differentiation of TH-IR amacrine cells, which serve as a model to gain insights into the mechanisms underlying developmental changes associated with the maturation of amacrine cells. Faintly staining TH-IR neurons are present in the proximal inner nuclear layer of newborn retinas. They are characterized by a large nucleus and usually a single primary process lacking varicosities. At postnatal day (PND) 6, TH-IR processes display more complex morphological characteristics, including a few varicosities, and second- and third-order ramifications. Growth cones are often seen. At PNDs 10 and 12 (eye opening), TH-IR cells have general morphological characteristics similar to adult TH-IR amacrines. They display 2-5 primary processes, which start forming a complex network of fibers in lamina 1 of the inner plexiform layer (IPL). TH-IR processes are also present in lamina 3 and rarely in lamina 5 of the IPL. Many fibers ending in growth cones are observed. In addition, very rare, thin TH-IR fibers are present in the outer plexiform layer. At PND 19, TH-IR fibers form a complex, dense network in lamina 1 of the IPL, and loose networks in laminae 3 and 5. Growth cones are not observed at this age. At PND 26, a few "ring-like" structures formed by TH-IR fibers in lamina 1 of the IPL are observed for the first time. In adult retinas, the "ring-like" structures are more numerous than at PND 26. A second, rare type of TH-IR cell ("type B") is encountered in all retinal regions beginning at PND 10. These cells are characterized by weak immunostaining and a small soma size. The present findings show that a significant differentiation of TH-IR neurons occurs during the first 10-12 PNDs. Eye opening is an important period for the maturation of TH-IR amacrines and, more generally, for the maturation of the IPL.     

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.     

Vasoactive intestinal polypeptide-containing cells in the rabbit retina: immunohistochemical localization and quantitative analysis

Vasoactive intestinal polypeptide (VIP) possesses neuroactive properties in the nervous system. In this study we characterized VIP immunoreactive neurons in the rabbit retina to provide a basis for a better understanding of the role of this peptide in retinal functions and to further define the morphology of wide-field amacrine cells. VIP immunoreactivity was demonstrated in colchicine-treated retinas. Immunolabeling was observed in amacrine cells located in the proximal inner nuclear layer and, occasionally, in the ganglion cell layer and inner plexiform layer (IPL). Varicose fibers were distributed in laminae 1, 3, and 5 of the IPL. The distribution of VIP immunoreactive cells showed a peak of approximately 50 cells/mm2 in the visual streak and minimum values of approximately 12 cells/mm2 in the peripheral retina. The total number of VIP immunopositive neurons was estimated to be about 11,000. Cell body diameters in the visual streak (8-9 microns) were slightly smaller than those measured in the dorsal or in the ventral retina (9-10 microns). The distribution of nearest neighbor distances (approximately 109 microns in the visual streak and approximately 99 microns in the peripheral retina) showed that VIP immunoreactive neurons were nonrandomly spaced. Labeled neurons emitted one to three thick primary processes, arborizing in secondary processes and collaterals rich in varicosities; these processes often crossed among different IPL laminae. Arborization fields of individual cells overlapped extensively. In the dorsal retina, estimated areas of single arborization fields were larger and processes had lower branching frequency than in the visual streak and in the ventral retina. On the whole, VIP immunoreactive amacrine cells gave rise to a loose meshwork of fibers in the IPL. These characteristics indicate VIP is contained in a class of wide-field amacrine cells and is likely to be involved in widespread regulatory or modulatory functions rather than in the direct transmission of visual information through the retina.